High-speed countercurrent chromatography (HSCCC) using a multilayer coiled column is a unique liquid-liquid partition technique that does not require the use of solid supports (Y. Ito, et al Crit. Rev. Anal. Chem., 17 65 (1986)). The use of two immiscible solvent phases in an open column free of solid support matrix can eliminate complications associated with conventional liquid chromatography such as tailing of solute peaks, adsorptive sample loss and deactivation, and sample contamination. On the other hand, counter current chromatography (CCC) separation often encounters difficulties for the stable continuous UV-monitoring of the effluent and, therefore, elution curves must usually be drawn manually by the spectrophotometric analysis of individual fractions after the effluent is fractionated with a fraction collector (Y. Ito, et al, J. Chromatogr., 244 247 (1982); T. Y. Zhang, et al, J. Chromatogr., 435 159 (1988); T. Y. Zhang, et al, Liq. Chromatogr., 11 233 (1988)). In order to avoid the above laborious procedure, it is highly desirous to establish a CCC monitoring system which produces stable UV-tracing of elution curves comparable to those in other chromatographic methods.
U.S. Pat. No. 4,019,372 to Parkell et al shows a liquid chromatography apparatus which uses a flow cell compartment mounted between a light source and a detector. The flow cell is transparent and is encapsulated in a thermally conductive medium for maintaining thermal equilibrium. The connecting tubing is in thermal contact with the flow cell for a sufficient length of time so that any liquid passing through it arrives to thermal equilibrium with the flow cell before entering the optically transparent flow cell. The effluent from the chromatographic column passes through tube which is partially coiled around the flow cell.
U.S. Pat. No. 4,051,025 is one of Dr. Ito's prior patents which is illustrative of an apparatus for which the present invention is intended. This patent shows a helical tube array rotating about its own axis in a gravitational field, where a countercurrent flow is produced by pumping a mobile phase through the rotating end of an helical array filled with a stationary phase. The mobile phase is finally eluted through the tail end of the tube. Any solutes introduced are subjected to a continuous partition process between mobile and stationary phases. At the outlet conduit there is an ultraviolet monitor to analyze the solute fractions. U.S. Pat. No. 4,487,693 is another example of a similar apparatus described by Dr. Ito.
U.S. Pat. No. 4,116,046 is cited to show a chromatography column with a temperature control jacket surrounding the column. This column and jacket are located prior to the detection device. The temperature control device is used to insure proper conditions for reproducible separations. A narrow bore tubing just downstream following the detector is used to establish back pressure and prevent bubbling.
U.S. Pat. No. 4,137,161 describes a liquid chromatographic apparatus having a flow restrictor downstream of the detector unit in order to apply back pressure to a single cell. The flow restrictor consists of a needle valve, a long thin capillary tube and a filter. This design prevents the formation of bubbles in the effluent from the separation column.
U S. Pat. No. 4,312,835 discloses a form of thermally controlling liquid chromatographic samples. The function of a temperature controlled platform is to uniformly maintain solution samples at a predetermined temperature until chromatographic analysis is completed. The patent states that this procedure is especially useful if the sample is labile and degrades at room temperature. The samples are positioned at 3, and the insulating layer is positioned at 2 shown in the figures. The sample holder body is made of a heat conductive material made of an inner surface and an outer surface. Pelteir elements comprise active surface 4a and reactive surface 4b and sample body surface 1b is in intimate contact with Pelteir active surface 4a. A heat exchanger is also placed in intimate contact with a Peltier surface 4b. Heating and cooling are a function of the active surface of the Peltier element.
U.S. Pat. No. 4,404,845 to Schrenker discloses a heat exchanger for the mobile phase and a separation column in a liquid chromatograph. The patent describes three methods of heat exchange. The third method includes a separation column which is concentrically mounted within a tube through which water flows. Control is maintained by means of a liquid convection thermal regulator. The purpose of temperature control is to avoid temperature gradients in the column and improve consistency of analysis as well as separation. FIG. 1 shows a capillary tube which leads to a heat exchange body. The mobile phase and the sample flow through this body to a separation column located in column compartment. In this patent heating or cooling is attained by heating or cooling the air surrounding the column.
U.S. Pat. No. 4,484,061 is another example of a temperature controlled system for liquid chromatographic columns where a foil-like patterned heating element is wrapped around the chromatographic column. A sensing element is also coextensively mounted with the heating element. The patent states that controlling the temperature of the column dramatically affects peak retention time reproducibility while maintaining a relatively constant base line or background electrical noise level due to solvent flow in the detector.
U.S. Pat. No. 4,814,089 shows the use of tapered restrictors and large interface heating zones for increasing pressure drop. It is also known to use thin walled capillary tubing to facilitate heat transfer. The patent also teaches that the mobile phase should not condense before detection as this would result in ion bursts which would produce extra electronic signals in the detector. While all of the comments in this patent relate to supercritical fluid chromatography and the problems associated with depressurization of the fluid stream, the teachings are not solely limited to this application.
Japanese Patent 1277-056 discloses a cylindrical heater for a chromatographic column arranged between the delivery part and the receiver.
The problems associated with the direct UV-monitoring of the effluent in CCC may be classified into the following four categories.
1) Steady carryover of the stationary phase due to an improper choice of the elution mode and/or the application of an excessively high flow rate of the mobile phase. PA1 2) Migration of the stationary phase into the flow cell which is caused by various conditions such as fluctuation of revolutional speed, vibration of the centrifuge system, and overloading of the sample, which may cause local alteration of the phase volume ratio and the physical properties of the two phases. PA1 3) Turbidity of a thermolabile mobile phase in the flow cell due to altered ambient temperature. PA1 4) Gas bubble formation in the effluent under reduced pressure in the periphery of the flow passage.
Of these, the first two problems may be avoided by choosing appropriate experimental conditions whereas solely a modification of the monitoring system has been found to overcome the latter two problems.
Accordingly, there is a definite need for an improved countercurrent chromatography apparatus which is capable of substantially eliminate turbidity in the flow cell caused by thermolabile components and the formation of gas bubbles in the effluent.